Download An artificial transcription activator mimics the genomewide

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reports
An artificial transcription activator mimics the
genome-wide properties of the yeast Pdr1
transcription factor
Frédéric Devaux, Philippe Marc, Céline Bouchoux, Thierry Delaveau, Immrich Hikkel,
Marie-Claude Potier1 & Claude Jacq+
Laboratoire de Génétique Moléculaire, CNRS UMR 8541, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris cedex 05 and 1Laboratoire de Neurobiologie et
Diversité Cellulaire, CNRS UMR 7637, Ecole Supérieure de Physique et de Chimie Industrielles de Paris, 10 rue Vauquelin, 75005 Paris, France
Received December 18, 2000; revised March 9, 2001; accepted April 26, 2001
We analysed the genome-wide regulatory properties of an
artificial transcription activator in which the DNA-binding
domain of the yeast transcription factor, Pdr1, was fused to
the activation domain of Gal4 (Pdr1*GAD). This Pdr1*GAD
chimera was put under the control of the inducible GAL1
promoter. DNA microarray analyses showed that all the target
genes upregulated by the well-studied native gain-of-function
Pdr1-3 mutant were similarly activated by the chimerical factor
Pdr1*GAD upon galactose induction. Additionally, this kinetic
approach led us not only to confirm previously published
targets, but also to define a hierarchy among members of the
Pdr1 regulon. Our observations prove, for the first time at the
complete genome level, that the DNA-binding domain of Pdr1
is sufficient to guide its specificity. We propose that this
approach could be useful for the study of new transcription
factors identified in silico from sequenced organisms. Complete
data are available at www.biologie.ens.fr/yeast-publi.html.
INTRODUCTION
The ability to specifically manipulate the expression of endogeneous genes or classes of genes would have wide-ranging
applications in medicine and in experimental and applied
biology. An efficient control of the genome expression processes
could be accomplished via the manipulation of transcription
factors and via an accurate understanding of gene regulation
which controls genome expression (Holstege and Young, 1999).
In that respect, transcription factor properties associated with the
DNA microarray technology can be regarded as precious tools
to decipher the intricacy of gene regulation networks. The aim of
this work was to assess the genome-wide regulatory properties
of a specific transcription factor DNA-binding domain, in order
to design a general strategy to reveal the target genes of new
yeast transcription factors in the absence of any other functional
information. For that purpose, the yeast transcription factor Pdr1
was taken as a model system. Pdr1 controls the transcription
of genes whose products are mainly involved in membrane
composition, such as the ABC transporter Pdr5 (Balzi, 1994),
and it plays a fundamental role in the pleiotropic drug resistance
phenomenon. A recent genome microarray analysis of a constitutively expressed PDR1 gain-of-function mutant (PDR1-3) has
shown that Pdr1 can upregulate 26 genes and downregulate 22
genes (DeRisi et al., 2000). However the interpretation of such
data can be obscured by the variety of secondary effects that might
be caused by a constitutive expression of a gain-of-function allele
(DeRisi et al., 2000). Moreover, the generalization of this kind of
approach is limited to transcription factors with known native
activated alleles, when in many cases only structural data based
on the sequence are available. With these problems in mind we
designed an experiment in which an artificially activated form of
Pdr1 was progressively produced under the control of the GAL1-10
promoter. Pdr1 has a modular structure typical of the 56 Gal4
family members (Johnston et al., 1986; Balzi et al., 1987): a short
N-terminal Zn(II)2Cys6 binuclear cluster DNA-binding domain
(100 amino acids) is linked to a C-terminal activation
domain (113 amino acids) via a rather long internal region
(668 amino acids) which contains multiple inhibitory domains
(Stone and Sadowski, 1993). Recently, a Gal4 version with a
+Corresponding author. Tel: +33 1 44 32 35 46; Fax: +33 1 44 32 39 41; E-mail: [email protected]
F. Devaux and P. Marc contributed equally to this work
© 2001 European Molecular Biology Organization
EMBO reports vol. 2 | no. 6 | pp 493–498 | 2001 493
scientific reports
F. Devaux et al.
large internal deletion was shown to possess all the known
properties of the full-length activated Gal4 protein (Ding and
Johnston, 1997). The rationale of our approach was to construct
an artificially active transcription factor containing only the
DNA-binding region from Pdr1, fused to the activated domain of
Gal4. We tested whether this chimerical construct could create
a Pdr1-specific transcription activator. Therefore, yeast microarray analysis was used to assess the quantitative differences
induced in the yeast transcriptome by a time course controlled
expression of this chimerical transcription factor. Our results are
consistent with previous studies of Pdr1 targets (DeRisi et al.,
2000) and they significantly improve our understanding of the
Pdr1 regulatory networks. They demonstrate that the Pdr1 DNAbinding domain alone is sufficient to confer the target gene
specificity of the native transcription factor. We thus suggest that
this strategy could be of general value to determine the function
of unknown transcription factors identified by genome sequencing.
RESULTS
Experimental strategy
We reasoned that hybrid transcriptional activators, composed of
a variable DNA-binding domain fused to a well known activation
domain like the Gal4-activation domain (GAD), could generate
a transcription activator reflecting the specificity of the DNAbinding domain. We then fused the DNA-binding domain of
Pdr1 (positions 1–207) to the Gal4-activation domain (positions
768–881) (Figure 1). This short chimerical protein, called
Pdr1*GAD, also contains the SV40 nuclear targeting signal,
three repeats of an HA epitope and the putative dimerization
domains of both Pdr1 and Gal4, as these factors are supposed to
act as homodimers. To control the production of Pdr1*GAD, the
chimerical gene was placed under the control of the GAL1-10
promoter in a centromeric plasmid (Figure 1A). The control
samples were conducted with the same plasmid expressing only
GAD. After galactose induction, the time course production of
the Pdr1*GAD protein was followed by gel electrophoresis
analysis (Figure 1B).
Physiological properties of the
chimerical activator Pdr1*GAD
We first verified that the production of Pdr1*GAD was conferring the same high level of drug resistance as the native gain-offunction mutant, Pdr1-3 (Carvajal et al., 1997), in cells lacking
both PDR1 and its functionally redundant homologue PDR3
(Figure 1C). This result suggests that the physiological activity of
the chimera is similar to that of the Pdr1-3 gain-of-function
mutant.
Global expression profile analysis of yeast cells
expressing the artificial Pdr1*GAD
transcriptional activator
We have used yeast whole-genome DNA microarrays to
compare the transcriptional properties of Pdr1-3 and Pdr1*GAD.
Expression profiles were generated in Saccharomyces cerevisiae
lacking PDR1 and PDR3. We first carried out five independent
494 EMBO reports vol. 2 | no. 6 | 2001
Fig. 1. Construction and properties of the chimerical transcription factor
Pdr1*GAD. (A) The DNA-binding domain of Pdr1 was fused to the C-terminal
domain of Gal4. The chimerical protein was designed to contain three N-terminal
HA epitopes and the SV40 nuclear targeting signal (NLS). This chimerical
gene was conditionally expressed under the control of the GAL1-10 promoter.
(B) Time course expression of the protein Pdr1*GAD after galactose
induction. The western blot shows two bands reduced to one single band, at
the expected size, after phosphatase treatment, suggesting that the protein can
be phosphorylated (data not shown). (C) Upon galactose induction, the
cycloheximide resistance phenotype is similar for yeast strains deleted for
both PDR1 and its functional homologue PDR3 (Δ1Δ3) expressing either the
Pdr1-3 gain-of-function or the Pdr1*GAD chimera.
DNA microarray experiments comparing the presence or
absence of the PDR1-3 gain-of-function allele. The results are in
general agreement with similar previous experiments (DeRisi et
al., 2000). However, repeat experiments with different sets of
DNA microarrays allowed us to obtain more accurate results. A
few genes: COS10, YGP1, HXK1, MET17, YGR243w,
YGR212w, YNR067c and YGL028c, which were previously
suggested as being upregulated in the presence of PDR1-3, did
not exhibit any significant expression changes in these new
scientific reports
An artificial transcription activator mimics yeast Pdr1
experiments. These transcripts have been shown to fluctuate
more than others in 63× repeats of a wild-type transcriptome
(Hughes et al., 2000), and they are probably false-positive candidates. On the other hand, a new set of genes was significantly
upregulated in the present study. These are FSP2, PDR10, RPN4,
YAL061w, YCR061w, YIl172c, YJL216c and YMR102c. PDR13-dependent upregulation of some of these genes was checked
by northern blot analyses (data on web site). Moreover, the fact
that all these genes have at least one pleiotropic drug responsive
element [PDRE, the binding site of Pdr1 as defined in DeRisi et
al. (2000)] in their promoter gives credence to these new findings. These experiments allowed us to define a new pattern of
Pdr1-3 activated targets, which was used as a reference to assess
the validity of the PDR1*GAD approach.
Microarray analyses of the time-controlled expression of
Pdr1*GAD protein, compared with a similar time course
production of GAD alone, reveal that the longest galactose timeinduction (18 h) actually mimics the constitutive production of
the Pdr1-3 gain-of-function transcription factor. A cluster
analysis of the upregulated genes revealed by the time course
experiments is presented in Figure 2. Simple visual comparisons
of data from Pdr1-3 (Figure 3A, left) and Pdr1*GAD (Figure 3A,
right) experiments reveal the strong similarity between the
transcriptional activity of the physiological gain-of-function
mutant and the artificial transcription factor. The large majority
of upregulated genes are common to both experiments. All but
one of these genes exhibit a PDRE in their promoter, and are
then strong candidates for a direct activation by the products of
both PDR1 alleles. Four genes contain PDRE and seem to be
specific for PDR1*GAD activation; two of them, HXT9 and
HXT11 (Nourani et al., 1997), are published targets of PDR1 and
the two others are their closest homologues: HXT8 and HXT12
(98% identities). We believe that this is an advantage of the
kinetic studies: to reveal genes that were not significantly
activated in a single condition experiment.
A more global comparison of Pdr1*GAD versus Pdr1-3
expression made by scatter plot did not reveal any striking differences between the two types of experiments (see Supplementary
data at EMBO reports Online).
Time course expression of the chimerical
transcription factor Pdr1*GAD reveals
waves of activated genes
Systematic studies of target genes associated with a transcription
factor are often limited by our ability to distinguish between
primary and secondary effects. We reasoned that a time course
production of the transcription factor should allow us to
distinguish stochastic fluctuations from biologically relevant
variations. In particular, the early activated genes are more likely
to correspond to direct targets of this transcription factor. These
early Pdr1 induced genes appear in the cluster analysis of
Figure 2. We analysed these data by principal components
analysis (PCA) (Figure 4). This representation discriminates the
specific transcriptional activations (Figure 4, right) or repressions
(Figure 4, left) by Pdr1-3 and Pdr1*GAD from the majority of
genes whose expression remains unchanged. It shows a gradient
of activated genes. However, we could not find a clear relationship between the number or the location of the PDRE in the
Fig. 2. Clustered display of upregulated genes from a time course production
of Pdr1*GAD. Cluster analyses have been conducted and represented as
indicated (Eisen et al., 1998). The first column represents the results obtained
when the Pdr1-3 gain-of-function mutant was constitutively expressed in a
strain deleted for both PDR1 and PDR3. The six subsequent columns present
the upregulated genes at different times after galactose induction of the
Pdr1*GAD construct (2, 4, 7, 10, 14, 18 h). All measurements are relative to
the GAD control taken at the same time of induction. Genes were selected if
their expression profile showed a continuous activation that goes beyond a 2-fold
induction factor for at least one time-point. The colour scale ranges are
indicated in the lower part. The early upregulated genes are presented in the
upper part; they all contain at least one PDRE in their promoter. The two last
lines correspond to ACT1 and PDA1 as internal controls, which are not
sensitive to the production of Pdr1*GAD. The complete set of data is available
at www.biologie.ens.fr/yeast-publi.html.
promoters and the kinetics of activation. Most of the genes that
are repressed by a constitutive expression of Pdr1-3 are similarly
downregulated by the chimera (see enhanced Supplementary
Figure at EMBO reports Online). Interestingly the repressed
EMBO reports vol. 2 | no. 6 | 2001 495
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F. Devaux et al.
Fig. 3. Comparison of the upregulated genes in PDR1-3 and PDR1*GAD
experiments. (A) Venn diagram showing the overlap between the PDR1-3 and
PDR1*GAD upregulated genes in microarray experiments, and the set of
yeast genes containing at least one Pdr1-binding site (PDRE) in their
promoter. The names of the corresponding genes are available on the web site
version of this figure (www.biologie.ens.fr/yeast-publi.html). Twenty-four genes
are upregulated by both proteins. 191 other genes which have a PDRE element
are neither regulated by Pdr1-3 nor by Pdr1*GAD. Respectively 2 and 3
PDRE-less genes are specific for PDR1-3 or PDR1*GAD expression. Four of
them (HXK1, INO1, URA1, YER067w) could account for differences in
culture and growth conditions between PDR1-3 and PDR1*GAD experiments
and might be considered as indirect effects. The last one (YOR152c) shares its
promoter with PDR5, the major target of Pdr1. (B) Promoter sequence
analysis of the 24 genes upregulated by either Pdr1-3 or Pdr1*GAD reveals a
core consensus sequence, which is TCCG(C/T)GGA. A systematic study of
the promoter of the 23 genes represented in Figure 2 led to a more precise
definition of the PDRE consensus. Note that the flanking regions are rather
pyrimidine rich on one side (left) and purine rich on the other side (right).
genes appear only at a later stage of induction of Pdr1*GAD,
suggesting that this repression is a secondary effect of Pdr1*GAD
overproduction. This is in agreement with the absence of PDRE
in their promoter sequences. We can not exclude the possibility
that Pdr1 has some repression effects, independent of its DNAbinding domain, but this point can not be assessed using the
Pdr1*GAD approach.
DISCUSSION
New insights in the PDR1 regulon
Time course expression of the chimerical Pdr1*GAD protein and
repetition of microarray experiments with the Pdr1-3 gain-offunction mutant allowed us to apply statistical methods to
analyse the data and to define a new set of Pdr1 upregulated
genes. In previous array experiments (DeRisi et al., 2000) the
26 genes that were shown to have increased expression in Pdr1-3
gain-of-function mutant were divided into five functional
groups: ABC transporters, major facilitator superfamily (MFS)
496 EMBO reports vol. 2 | no. 6 | 2001
Fig. 4. The microarray results were analysed by PCA (Dysik and Jonassen,
2001) for determining the two key variables represented on the two axes
(PCA1 versus PCA2). This analysis clearly distinguishes the group of
repressed genes (left, green) and the group of activated genes (right, red) from
the bulk of the genes (central, blue). More interestingly, the upregulated genes
are distributed along a gradient, which could reflect the existence of distinct
subgroups of genes (see text). The expression profiles of repressed (A) and
upregulated (B) genes are represented.
and other permeases, lipid metabolism, cell wall metabolism
and stress response. None of the genes of the stress response
group apart from one (GRE2) were significantly upregulated. Repetitions of experiments with the PDR1-3 mutant clearly showed
that these stress group genes are not directly upregulated by
Pdr1. This is supported by the fact that none of these genes have
a PDRE in their promoter sequence. On the other hand, new
Pdr1 target genes have been discovered. An interesting case is
that of SON1/RPN4. This gene has been described as coding for
a transcription regulator for genes encoding subunits of the
proteasome (Mannhaupt et al., 1999). We might suspect that
RPN4 was activated as a rather trivial response to Pdr1*GAD or
Pdr1-3 overproduction. This is unlikely since we never observed
such a signal with other transcription factor*GAD fusions (data
not shown). Moreover, two PDRE elements are localized in the
RPN4 promoter and the connection recently established
between RPN4 and the yeast response to alkylating agents
strengthens the idea that the proteasome function and the
pleiotropic drug resistance phenomenon could be linked
(Jelinsky et al., 2000).
A general strategy to decipher the genomic
signature of a transcription factor
It is known that a given transcriptional regulator may activate
transcription of one gene, repress transcription of another and
bind but exert no regulatory effect on a third (Lefstin and
Yamamoto, 1998). This transcriptional flexibility is probably in
part controlled by the central tethering region of factors like the
Gal4 family members (Carvajal et al., 1997), and also by
functional interactions between DNA-binding domains and
activation domains as described for the USF2 transcription factor
(Luo and Sawadogo, 1996). In such a context, the present
domain-swap experiment in which the DNA-binding and dimer-
scientific reports
An artificial transcription activator mimics yeast Pdr1
ization domain of Pdr1 is directly linked to the Gal4 activation
domain may seem naïve. We demonstrate, however, that this
chimerical transcriptional activator Pdr1*GAD generates a
transcriptome profile very similar to that of the ‘natural’ Pdr1-3
gain-of-function mutant. These two forms of Pdr1, which share
only the Pdr1 DNA-binding domain, upregulate the same set of
23 PDRE-containing genes (Figure 3A). This implies that the Pdr1
DNA-binding domain discriminates faithfully these 23 genes
among the 218 PDRE-containing genes of the yeast genome. For
the moment, we did not find clear differences between the
consensus sequence defined from these 23 regulated genes
(Figure 3B) and the PDRE found in the promoters of the nonregulated genes. This strongly suggests that the Pdr1 DNA-binding
domain harbours activities beyond simple DNA recognition, a
point which is supported by recent observations on several transcription factors (Lefstin and Yamamoto, 1998). We have
evidence that such an approach could be efficient for other
S. cerevisiae Gal4 family members (work in progress) and thus
may be extended to similar transcription factors in other eukaryotes
and pathogenic yeasts. Moreover, it might be extended to other
classes of transcription factors, provided that their DNA-binding
domain can be clearly defined and that they do not need any
cofactor for their target specificity.
The principal objective of this work was to define a gene target
search based only on the available transcription factor structural
data. We state that, in the case of Pdr1, the knowledge of the
DNA-binding domain is necessary and sufficient to define the
corresponding pattern of regulation. It would not be correct to
claim that such a pattern of regulation reflects all the properties
of the transcription factor. The regulation pattern that we found for
both the chimerical Pdr1*GAD and the Pdr1-3 gain-of-function
mutant reflects a similar derepressed state of the protein. In that
respect the upregulated genes represent the wide regulatory
network of Pdr1. We propose to call this set of genes the
genomic signature of the transcription factor. This genomic
signature can be connected to other gene expression groups
recently identified (Hughes et al., 2000). For instance, PDR15,
SNQ2, YOR1, YAL061w, YMR102c, YOR049c belong to the
same functional cluster and are an important part of the Pdr1
regulon. The Pdr1 genomic signature can thus be considered as
composed of several functionally related groups of genes.
Kinetic studies and PCA (Figure 4) can also distinguish gene
subgroups inside this genomic signature.
In conclusion, we believe that the systematic analyses of
genomic signatures of known or unknown transcription factors,
combined with more physiological approaches such as the
compendium, should be an important asset in understanding the
global functional properties of a genome. The approach
described in this work should help in reaching this goal in yeast,
and may be extrapolated to any sequenced organism.
cloned at the XhoI site of the plasmid pCB by homologous
recombination in S. cerevisiae. Plasmid pCB-PDR1*GAD was
constructed by insertion of the 5′ 620 first nucleotides of the
PDR1 open reading frame in the pCB-GAD plasmid. This
sequence was amplified by PCR and cloned at the NotII site of the
pCB-GAD plasmid by homologous recombination. For the gain-offunction mutant experiments, we used the centromeric pRS315
vector containing the PDR1-3 gain-of-function allele of PDR1
under the control of its own promoter (Carvajal et al., 1997). The
empty pRS315 was used as a control.
Strains. The yeast strains used in this study were isogenic to
FY1679-28C Δpdr3 Δpdr1 (Mata ura3-52 trp1Δ63 leu2Δ1
his3Δ200 Δpdr3 ::HIS3 Δpdr1 ::TRP1).
Cell cultures. FY Δpdr3 Δpdr1 cells in exponential growth,
containing either the pCB-PDR1*GAD or the pCB-GAD vectors
were shifted from glucose to galactose minimal media (2%
sugar, 0.67% yeast nitrogen base without amino acids, supplemented with adenine and leucine). Cells were then collected at
different times (from 0 to 18 h) after medium change for RNA
extraction and microarray analyses. FY Δpdr3 Δpdr1 cells
containing either the pRS315-PDR1-3 or the pRS315 vectors
were grown in glucose minimal media supplemented with
adenine and uracil to an OD of 0.7–0.8.
Microarray analyses. The microarrays containing all the yeast
open reading frames were obtained from the Toronto microarray
center and from Hitachi Software. They were based on the principle of PCR products deposited onto a polyamine coated glass
slide (Eisen and Brown, 1999). All experiments were performed
at least twice except for times 2 and 4 h, which were performed
only once. The detailed microarray protocols are available at our
web site. A 2 μg aliquot of mRNA was used for each reverse transcription. The arrays were read using a genepix 4000 scanner from
Axon and analyzed with the genepix 3.0 software.
Bioinformatical analyses. We filtered data, excluding artefactual
spots, saturated spots and low signal spots. Assuming that most
of the genes have unchanged expression, the Cy3/Cy5 ratios
were normalized using the median of all the ratios for each
experiment. We clustered the data from 12 independent experiments using PCA module of J-express (Dysik and Jonassen,
2001). The profiles of genes belonging to up- and downregulated clusters were visually checked and genes with nonregular profiles were discarded. For motif search in the promoter
(between –800 and +1) of the upregulated genes we used the
Consensus (van Helden et al., 1998) module of RSA tools (van
Helden et al., 2000). The consensus sequences found were then
aligned and represented using Sequence logo (Schneider and
Stephens, 1990). The cluster of Figure 2 was generated using
Treeview (Eisen et al., 1998).
Supplementary data. Supplementary data are available at EMBO
reports Online and www.biologie.ens.fr/yeast-publi.html.
METHODS
ACKNOWLEDGEMENTS
Plasmids. Plasmid pCB-GAD was obtained from the plasmid
pCB [derived from pYES2 (Invitrogen): URA3, AmpR, pMB1 ori,
ARS4-CEN6 ori, GAL1 promoter, three HA epitopes and CYC1
terminator] as follows: ‘SV40 NLS/Gal4 activation domain’
sequence was amplified by PCR from the two-hybrid plasmid
pACT2 (see oligonucleotide sequences at: www.biologie.ens.fr/fr/
genetiqu/puces/pucesadnframe.html). This PCR product was
We are especially grateful to Andre Goffeau for continuous
encouragement and for critical reading of the manuscript. We
thank John Friesen for his advice and Elvira Carvajal, Sylvie
Hermann, Stephane Le Crom and Marisol Corral-Debrinski for
their comments on the manuscript. Thank you to Julian Ghislain
for grammatical corrections. We are grateful to Hitachi Software
and to the Toronto Microarray Center for providing yeast
EMBO reports vol. 2 | no. 6 | 2001 497
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F. Devaux et al.
microarrays. This study was supported by the Association pour
la Recherche contre le Cancer (ARC No. 5691) and by MENRT.
REFERENCES
Balzi, E., Chen, W., Ulaszewski, S., Capieaux, E. and Goffeau, A. (1987) The
multidrug resistance gene PDR1 from Saccharomyces cerevisiae. J. Biol.
Chem., 262, 16871–16879.
Balzi, E., Wang, M., Leterme, S., VanDyck, L. and Goffeau, A. (1994) PDR5,
a novel yeast multidrug resistance conferring transporter controlled by the
transcription regulator PDR1. J. Biol. Chem., 269, 2206–2214.
Carvajal, E., Van den Hazel, H.B., Cybularz-Kolackowska, A., Balzi, E. and
Goffeau, A. (1997) Molecular and phenotypic characterization of yeast
PDR1 mutants that show hyperactive transcription of various ABC
multidrug transporter genes. Mol. Gen. Genet., 256, 406–415.
DeRisi, J., Van Den Hazel, B., Marc, P., Balzi, E., Brown, P.O., Jacq, C. and
Goffeau, A. (2000) Genome microarray analysis of transcriptional
activation in multidrug resistance yeast mutants. FEBS Lett., 470, 156–160.
Ding, W. and Johnston, S. (1997) The DNA binding and activation domains
of Gal4p are sufficient for conveying its regulatory signals. Mol. Cell.
Biol., 17, 2538–2549.
Dysik, B. and Jonassen, I. (2001) J-express: an interactive Java based tool for
exploration of gene expression data sequences. Bioinformatics, 17, 369–370.
Eisen, M.B. and Brown, P.O. (1999) DNA arrays for analysis of gene
expression. Methods Enzymol., 303, 179–205.
Eisen, M.B., Spellman, P.T., Brown, P.O. and Botstein, D. (1998) Cluster
analysis and display of genome-wide expression patterns. Proc. Natl
Acad. Sci. USA, 95, 14863–14868.
Holstege, F.C.P. and Young, R.A. (1999) Transcriptional regulation:
contending with complexity. Proc. Natl Acad. Sci. USA, 96, 2–4.
Hughes, T. et al. (2000) Functional discovery via a compendium of
expression profiles. Cell, 102, 109–126.
498 EMBO reports vol. 2 | no. 6 | 2001
Jelinsky, S., Estep, P., Church, G. and Samson, L. (2000) Regulatory
networks revealed by transcriptional profiling of damaged
Saccharomyces cerevisiae cells: Rpn4 link base excision repair with
proteasomes. Mol. Cell. Biol., 20, 8157–8167.
Johnston, S., Zavortink, M., Debouck, C. and Hopper, J. (1986) Functional
domains of the yeast regulatory protein GAL4. Proc. Natl Acad. Sci. USA,
83, 6553–6557.
Lefstin, J. and Yamamoto, K. (1998) Allosteric effects of DNA on
transcriptional regulators. Nature, 392, 885–888.
Luo, X. and Sawadogo, M. (1996) Functional domains of the transcription
factor USF2: atypical nuclear localization signals and context-dependent
transcriptional activation. Mol. Cell. Biol., 16, 1367–1375.
Mannhaupt, G., Schnall, R., Karpov, V., Vetter, I. and Feldmann, H. (1999)
Rpn4p acts as a transcription factor by binding to PACE, a nonamer box
found upstream of 26S proteasomal and other genes in yeast. FEBS Lett.,
450, 27–34.
Nourani, A., Wesolowski-Louvel, M., Delaveau, T., Jacq, C. and Delahodde, A.
(1997) Multi-drug-resistance phenomenon in the yeast Saccharomyces
cerevisiae: involvement of two hexose transporters. Mol. Cell. Biol., 17,
5453–5460.
Schneider, T. and Stephens, R. (1990) Sequence logos: a new way to display
consensus sequences. Nucleic Acids Res., 18, 6097–6100.
Stone, G. and Sadowski, I. (1993) GAL4 is regulated by a glucose-responsive
functional domain. EMBO J., 12, 1375–1385.
van Helden, J., Andre, B. and Collado-Vides, J. (1998) Extracting regulatory
sites from the upstream region of yeast genes by computational analysis of
oligonucleotide frequencies. J. Mol. Biol., 281, 827–842.
van Helden, J., Andre, B. and Collado-Vides, J. (2000) A web site for the
computational analysis of yeast regulatory sequences. Yeast, 16, 177–187.
DOI: 10.1093/embo-reports/kve114